A new locus for autosomal recessive congenital cataract identified in a Pakistani family.

Purpose To identify the disease locus for autosomal recessive congenital cataract in a consanguineous Pakistani family. Methods All affected individuals underwent detailed ophthalmologic and medical examination. Blood samples were collected and DNA was extracted. A genome-wide scan was performed with polymorphic microsatellite markers on genomic DNA from affected and unaffected family members, and logarithm of odds (LOD) scores were calculated. Results The clinical records and ophthalmological examinations suggested that all affected individuals have nuclear cataracts. Maximum LOD scores of 5.01, 4.38, and 4.17 at θ=0 were obtained with markers D7630, D7S657, and D7S515, respectively. Fine mapping refined the critical interval and suggested that markers in a 27.78 cM (27.96 Mb) interval are flanked by markers D7S660 and D7S799, which co-segregate with the disease phenotype in family PKCC108. Conclusions We have identified a new locus for autosomal recessive congenital cataract, localized to chromosome 7q21.11-q31.1 in a consanguineous Pakistani family.

Congenital cataracts are one of the major causes of vision loss in children worldwide and are responsible for about onethird of cases of blindness in infants [1,2]. They can occur in an isolated fashion or as one component of a syndrome affecting multiple tissues. Nonsyndromic congenital cataracts have an estimated frequency of 1-6 per 10,000 live births [3]. They can lead to permanent blindness by interfering with the sharp focus of light on the retina, especially during the early developmental periods. Morphologically, different types of cataract are classified according to the part of the opacified lens, including nuclear, cortical, lamellar, sutural, polar, or subcapsular cataract [4].
Mutation screening: Primer pairs for individual exons were designed using the primer3 program. The sequences, and annealing temperatures, and product size are shown in Table  1. Amplifications were performed in 25 μl reaction volume containing 50 ng of genomic DNA, 400 nM of each primer, 250 μM of dNTPs, 2.5mM MgCl2, and 0.2 U Taq DNA polymerase in the standard PCR buffer provided by the manufacturer (Applied Biosystems). PCR amplification consisted of a denaturation step at 96 °C for 5 min followed by 40 cycles, each consisting of 96 °C for 45 s followed by 57 °C for 45 s and 72 °C for 1 min. PCR products were analyzed on 2% agarose gel and purified by ethanol precipitation. The PCR primers for each exon were used for bidirectional sequencing using BigDye Terminator Ready reaction mix, according to manufacturer instructions. Sequencing products were precipitated and resuspended in 10 μl of formamide (Applied Biosystems) and denatured at 95 °C for 5 min. Sequencing was performed on an ABI PRISM 3100 Automated sequencer (Applied Biosystems). Sequencing results were assembled with ABI PRISM

RESULTS
The family described in this study, PKCC108 (Figure 1 [7], we therefore sequenced all coding exons, exon-intron boundaries and the 5′ untranslated region of GJE1 but did not identify any pathogenic mutations.

DISCUSSION
Here we report identification of a new locus for autosomal recessive cataract mapped to chromosome 7q21.11-q31.1 in a consanguineous Pakistani family (PKCC108). A maximum LOD score of 5.08 was obtained with D7S2540 at θ=0, and calculated using ILINK. Asterisk: STR Marker includeed in genome wide scan. the disease locus co-segregates with chromosome 7q markers in 27.78 cM (27.96 Mb) interval flanked by D7S660 and D7S799. The lack of significant LOD scores other than those in the chromosome 7q region during the genome-wide linkage scan, homozygous alleles for all the affected individuals for markers of 7q, and lack of homozygosity in all the unaffected individuals strongly suggest that the disease locus maps to a new region on chromosome 7q. No previously reported cataract loci or genes are located in the critical interval.
To date, 12 loci for recessive congenital cataract have been identified, illustrating the genetic heterogeneity of the disease. Previously, nuclear cataracts segregating in consanguineous families have been mapped to chromosome 2p12, 19q13.4, and 22q12.3, in the latter of which mutations in CRYBB3 were identified [12,16,21]. All of these families are of Pakistani origin, suggesting that nuclear cataract is prevalent in the Pakistani population.
The critical interval harbors GJE1, a gene that is a member of the gap junction protein family. As the lens is an avascular structure that lacks almost all cell organelles, cellcell communication is brought about by an extensive system of gap junction proteins. Gap junction proteins GJA1 (connexin 43), GJA3 (connexin 46), and GJA8 (connexin 50) are expressed in human lens, and mutations in GJA3 have been reported to cause cataract with different morphology [22,23]. Similarly, there are many reports of mutations in GJA8 [24,25]. We sequenced all coding exons, exon-intron boundaries, and the 5′ untranslated region of GJE1 but found no pathogenic mutations.
Transparency and precise shape and optical density are distinctive features of the lens that are critical for proper light refraction. Elucidating the molecular mechanisms that maintain or disrupt lens transparency is a fundamental step toward preventing cataract. Identification of new genes involved in cataract will help us understand the molecular biology of the human lens and the structural and metabolic mechanisms involved in maintenance of the transparency of the lens.